The results of the present study further indicated the molecular mechanism of Lp299v supplementation. Meanwhile, the core genes and the results of enrichment analyses expanded the molecular mechanism of Lp299v supplementation in patients with stable CAD. Our experimental data were categorized into the DAU group and the non-DAU group. The former was composed of 8 samples, and the latter included 30 samples. The bioinformatics analysis was performed on both the DAU and the non-DAU groups. After the results were compared between the two groups, some differences were found, which could reveal the weakening effect of alcohol on the protection of intestinal inflammation. This hypothesis will be further analyzed in the future experiments.
The composition of gut microbiota is very complex, including bacteroidetes, firmicutes, proteobacteria, fusobacteria, actinobacteria. In different populations with different diets, gut microbiota vary in terms of number and proportion (Yamashita et al. 2016). Previous studies have found that gut microbiota were associated with several diseases, such as cardio-metabolic diseases, tumors, inflammatory bowel disease, and central nervous system diseases (Cheng et al 2020; Sitkin et al. 2021; Sun et al. 2020; Zhou et al. 2020). Some scholars demonstrated that intestinal inflammation may be a risk factor for atherosclerosis, and gut microbiota and intestinal immunity could be used as therapeutic targets for the treatment of CAD (Yamashita et al. 2015). Lactobacillus plantarum is one of the important members of the genus Lactobacillus, and it has been identified as a probiotic, confirming its value for further research and application (Kleerebezem et al. 2010).
In both the DAU and non-DAU groups, the GO enrichment analysis indicated cell mitosis and reduced chemotactic movement, suggesting that Lp299v could promote immune regulation and attenuate immune response. Additionally, the GO enrichment analysis revealed that cell mitosis, anti-apoptosis and anti-cell senescence increased, which may be secondary to the activation of the PI3K-Akt pathway. The KEGG pathway enrichment analysis showed apoptosis and senescence of cells in the DAU group, as well as the down-regulated expression of HSV-1 infection-related signaling pathway, while in the non-DAU group, neuroactive ligand-receptor interaction and various infection-related signaling pathways were up-regulated to varying degrees. In the DAU group, activation of these pathways inhibited cellular senescence and apoptosis, attenuated innate immune escape, and reduced inflammatory response. In the non-DAU group, activation of these pathways inhibited the proliferation and recruitment of macrophages, decreased the expression of interferon-β (INF-β), and attenuated immune response and chemotactic cytokines. A previous study showed that alcohol intake could promote the growth of Gram-negative bacteria in the intestines and increase the permeability of the intestines, leading to systemic inflammations (Parlesak et al. 2000). Another study which included in vivo and in vitro experiments showed that Lp299v improved vascular endothelium. The possible mechanisms might be related to improved endothelium-dependent vasodilation and nitric oxide (NO) bioavailability, as well as reduced levels of IL-8, IL-12, and leptin (Malik et al. 2018). It may be because leptin may activate p38 and ERK signaling pathways by interacting with a leptin receptor, ObR, to induce IL-8 production in M2 macrophages (Cao et al. 2016). A previous clinical study showed that in patients with obstructive jaundice, Lp299v supplementation after biliary drainage could reduce acute inflammation of the immune system and decrease intestinal permeability (Jones et al. 2013). A number of scholars confirmed that Lp299v could attenuate the immune response in patients with coronary heart disease (Hofeld et al. 2021; Naruszewicz et al. 2002). A previous study reported that Lp299v significantly reduced cell apoptosis, which was consistent with the down-regulation of apoptosis pathway found in our study (Dykstra et al. 2011). Earlier studies showed that Lp299v-contained beverages could protect body cells against excessive production of reactive oxygen species, thereby protecting against oxidative damage (Gawlik-Dziki et al. 2021; Onning et al. 2003).
We, in the present study, found genes that were associated with G protein-coupled receptor, inflammatory response, cell cycle regulation, regulation of cell proliferation and apoptosis, and metastasis suppression in the PPI networks. A previous study reported that Lp299v could be used in the treatment of some types of cancer, possibly because Lp299v affects cell cycle, cell proliferation and apoptosis, tumor metastasis, inflammatory response, and other genes and proteins (Kazmierczak-Siedlecka et al. 2020). In our screening of core genes, it was found that the majority of the core genes were G protein-coupled receptor-related genes, followed by inflammation-activated chemotaxis-related genes. Diverse types of chemokines act through G protein-coupled receptors, which are collectively known as chemokine receptors. Interleukin receptor and histamine receptor are involved in inflammation and allergic reactions. In the current study, it was hypothesized that one or more of the components of Lp299v could modulate the G protein-coupled receptor, thereby regulating the immune system, inhibiting inflammatory response, promoting cell mitosis and proliferation, and inhibiting cellular senescence and apoptosis.
We found the expressions of the genes encoding adenylate cyclase, and β- and γ-subunit of G protein-coupled receptor. The results of enriched MF of DEGs indicated that chemokine receptors, acting as a G protein-coupled receptor, could play important roles in the immune system cell signaling pathway. Using the constructed PPI networks, it was found that the expressions of CXCL11 and CXCR3 in the DEGs of this sample had a parallel relationship. Both CXCL11 and CXCR3 are pro-inflammatory factors. In patients with irritable bowel syndrome, the mRNA levels of IL-6, CXCL-11, and CCR3 were up-regulated (Shukla et al. 2018). A recent study showed that compared with CCl4-treated mice that did not receive probiotic strains, mice that received B. pseudocatenulatum CECT7765 had a reduced inflammatory environment and bacterial translocation (Moratalla et al. 2014). In another study, the expression levels of pro-inflammatory factors (CCR6, CCR9, CXCR3, and CXCR6) were reduced in intestinal lymphocytes in mice with liver cirrhosis taking Bifidobacterium pseudocatenulatum CECT7765 compared with the control group (Moratalla et al. 2016). Hence, it was further hypothesized that Lp299v supplementation could modulate the expressions of chemokine receptors via a subsequent cellular effect.
In summary, Lp299v could treat patients with stable CAD by modulating inflammatory responses. Our study also found the role of G protein-coupled receptor, mitosis, apoptosis, and senescence of cell in the process of Lp299v supplementation. Cell mitosis, apoptosis, and senescence were associated with the PI3K-Akt pathway. The chemokine receptors could act as a G protein-coupled receptor, playing important roles in the immune system cell signaling pathway. It was supposed that chemokine receptors could have a cellular effect through the Gs-cAMP-PKA signaling pathway.
In this study, we found the following limitations. First, the small sample size might cause the bias in the experiment. Second, several core genes and molecular pathways were identified, which were correlated with the effect of Lp299v supplementation, while we did not explore the interactions among these core genes. Finally, we did not determine which type of chemokine receptors and which ligand play regulatory roles. Thus, our findings remain to be further verified by additional in vivo or in vitro experiments.